Apparatus for transferring separate liquid fractions into separate fraction vessels

ABSTRACT

Apparatus for transferring a plurality of liquid fractions having differing characteristics discharged from a single source which includes a multipath valve connected between the source and a series of receptacles, with a controller which senses the liquid discharged and operates the valve to direct the various fractions into the receptacles in accordance with the characteristics of the fraction being discharged.

United. States Patent Keck [4 Oct. 16, 1973 1 APPARATUS FOR TRANSFERRING SEPARATE LIQUID FRACTIONS INTO SEPARATE FRACTION VESSELS [76] Inventor: Klaus Keck, 775-Konstanz, Jacob Burckhardt-Str. l4, Constance, Germany 22 Filed: Mar. 3, 1972 21 Appl. No.: 231,688

[52] US. Cl 141/234, 23/253, 23/259,

137/625.l1, 222/52 [51] Int. Cl B65b 3/04 [58] Field of Search 141/99, 234-248,

[56] References Cited UNITED STATES PATENTS 3,319,655 5/1967 Palmer 137/62511 7. Z %/J g K PH. KB D\ 3,678,959 7/1972 Liposky 137/625.11

Primary E xaminerl-louston S. Bell, Jr. Attorney-Arnold B. Christen et a1.

[57] ABSTRACT Apparatus for transferring a plurality of liquid fractions having differing characteristics discharged from .a single source which includes a multipath valve connected between the source and a series of receptacles, with a controller which senses the liquid discharged and Operates the valve to direct the various fractions into the receptacles in accordance with the characteristics of the'fraction being discharged.

1 Claim, 9 Drawing Figures PATENTEDUCT 16 1915 SHEET 1 UF 4 Fi defghi k PATENTEUUCT 16 I975 SHEET 3 [1F 4 Fig.6

APPARATUS FOR TRANSFERRING SEPARATE LIQUID FRACTIONS INTO SEPARATE FRACTION VESSELS This invention refers to an apparatus for handling fractions separated e.g. by means of column chromatography wherein the sample tube separated is supplied to a generally vertical column which is packed with a stationary medium, which considerably reduces the velocity of the flow through the column, the fractions of the sample being discharged sequentially from the bottom of the column. More specifically the invention refers to an apparatus for transferring the separated liquid fractions to different fraction vessels.

Separation of substances for preparative and analytical purposes are very common in the chemical and biological fields. A widely used method for such separation is column chromatography wherein a suitable stabilizing medium could be chosen with respect to phase and chemical composition of the substances to be separated. Thus ion exchangers, molecular sieving compounds, aluminium oxide, cellulose, carbon etc. could be used as a stationary phase, the substances to be separated interacting differently with respect to chemical or physical properties with the stationary medium.

In most prior art devices in this field the substances discharged sequentially from the column are transferred to small vessels e.g. test tubes for collection and are then subject to further analysis or reactions. The test tubes, generally between 50 200, are arranged in racks of a fractioncollector which stepwise advances the racks. This stepwise moving is usually controlled by the discharged volumes from the column, e.g. by having a counter that counts the drops that leave the column and generates a trigger impulse to the rack moving mechanism when a certain number of drops has been collected in a tube. The racks could also be advanced at definite time intervals, the length of which is calculated from the drop rate of the column.

The liquid is usually discharged from the column to the fraction collector via a detector, by means of which the different separated fractions could be detected, this detector being connected to some signal registering device, e.g. a plotter, so that'when the discharge is completed, a curve indicating the contents of the different test tubes is obtained.

The detector might e.g. consist of a UV-light source, the light of which impinges on the discharged sub:

stance, the transmitted light being detected by a photometer. Such a detector could be used when the fractions differ with respect to UV-absorption. Also visible light could be used. Other types of detectors are e.g. pl-l-meters, electrical conductivity meters, heat conductivity meters or other thermic meters. Furthermore, the radioactive emission from the fractions could be detected. The output signal from the detector could also be used for controlling the movements of the fraction collector. Thus the US. Pat. No. 3,202,183 describes a system where the racks are advanced each time the derivative of the detection signal becomes positive, thus discharging each fraction into a separate sequence of test tubes.

The main disadvantages of the above described collecting apparatus could be listed as follows:

In many separations of liquid samples one is only interested in some fractions in a sample of a known composition for further treatment whereas the major part of the separated sample is of no interest. However, most commercially available fraction collectors have a very high number of small test tubes and a high number of test tubes will be wasted in each fraction collector.

Furthermore, certain fractions have to be protected immediately after separation by certain agents, (e.g. mercaptane or sterilizing agents, protecting gas, etc.) In order to make this in the above described apparatus such agents must be added to several hundred test tubes, as it will not be possible to predict the tubes where the fractions of interest will be collected. It is an object of the present invention to provide an improved apparatus for transferring liquid fractions into separate fraction vessels, the improvement consisting therein that each detected and registered fraction of interest is transferred to one collecting vessel irrespective of its specific volume, all remaining fractions being collected in a common waste vessel.

Preferred embodiments of the invention will now be described in detail with reference to the accompanying drawing in which:

' FIG. 1 schematically shows a prior art system for collecting the fractions from a column chromatograph;

FIG. 2 is a characteristic of fractions discharged from a column chromatograph;

FIG. 3 shows partly in section the mechanical part of a fraction distributing valve of the invention;

FIG. 4 shows a cross sectional view along line CD in FIG. 3;

FIG. 5 shows a cross sectional view along line EF in FIG. 3;

FIG. 6 shows a cross sectional view along line GH in FIG. 3; and

FIG. 7 is a circuit diagram of the control means of the fraction distributor of the invention.

Referring now to FIG. 1 there is shown schematically a prior art column chromatography system. In this system the compound to be separated is supplied, usually in liquid phase, to the column S containing a stationary phase, e.g. an ion exchanger J. If a discharging compound L is supplied to the column S, the separated substances will be washed out from the ion exchanger. The sequence and velocity of the washed-out separated substances will then be dependent upon the coupling of the different fractions to the stationary phase. The volume of the discharging compound is usually much higher than the volume of the column and thus the washed out substances will be highly diluted.

The substances E discharged at the bottom of the column S will flow or drop into a line of test tubes or similar small containers KB, which are changed when they are filled up to a certain degree, so that when the discharge is completed, a number of containers will be obtained each containing an equal amount of the discharged substance E.

When leaving the column, the substance E passes a cuvette K arranged at the bottom of the column. In the apparatus according to FIG. 1 the fractions are supposed to differ with respect to their'UV-absorption. The detector consequently consists of a Uv light source from which UV-light is transmitted through the cuvette to a photometer PH from which a signal corresponding to the UV-absorption of the sample is transferred to a diagram D of a plotter.

When the separation is completed, the strip D will be provided with a diagram in which the abscissa represents time whereas the ordinate represents the correpoints of time when a new test tube is moved to receive the substances discharged from the column. As can be seen from the curve, the fraction F4 will e.g. be supplied to the tubes e-h, fraction F5 to the tubes k-m and fraction F6 to the tubes n-o. Thus the tubes e, k,'m, and will contain a mixture of subsequent fractions and the contents of these containers usually cannot be used. For further analysis or treatment only the test tubes I and m for the fractions F and F6 respectively, can be used, whereas the useful part of fraction F4 is distributed in four containers,f, g, h, and i.

In the apparatus of the invention the collecting vessels will be changed at each minimum point of the curve, i.e. at the borderlines between subsequent fractions. This means that each of the fractions F l-F7 will be discharged in a separate vessel. Of course one will in practice obtain a certain mixing of the fractions in the containers (asindicated by the dotted line between F2 and F3) so that e.g. the vessel for the fraction F2 will also comprise small amounts of the fraction F3. This effect will, however, also be present in the hitherto used methods and could be eliminated by repeated fractioning of the sample. In most cases this effect could, however, be neglected, and the result of the separation according to the invention could be directly used for further analysis.

To ensure that the change of containers will take place at the change of fractions, the apparatus according to the invention is provided with a control unit, connected to the detection unit, this control unit comparing each sample detecting signal with the previous sample. FIG. 2A shows a part of a fraction characteristic, the difference between subsequent sample values being indicated.

In FIG. 2A the time axis runs from left to right and thus the values U2, U2, denote the later detection signals (according to the example the voltage or current values obtained from the photometer PH) and the values U1, U1 etc. denote the previous sample values. If the later values are compared with the previous values, the sign of the difference indicates if the fraction characteristic is rising or falling.

The control unit connected to the detector indicates the polarity of the differences AU, AU, etc. and according to the invention these signals are used to generate a trigger impulse, when the polarity changes from a negative to a positive value, whereas no trigger signal is generated if the polarity changes from a positive to a negative value.

A device for this signal analysis could easily be made from well-known electronic components.

According to the invention the trigger impulses control a multipath valve, having an input port connected to the column, and a number of different output ports connected to funnels of different containers.

An embodiment of such an apparatus is shown in FIGS. 3-6. In this apparatus a flexible tube 1 is connected to the detecting means EA, this tube terminating in a plug 2 of a multipath valve .3 provided with a first group of output ports 4. Each port 4 is via a flexible tubing 5 connected to a vessel 6.

The plug 2 is e.g. over a stem 7 connected to a driving device, the plug being moved from one output port to the following each time the stem is operated. The stem- '7 is provided with threads and terminate in a nut in such a way that when the stem is rotated one half turn, the plug is moved from one output port to the following. Of course other types of multipath valves could also be used, e.g. multipath valves having output ports distributed along the periphery of a circle etc.

The stem 7 is provided with a threaded groove 8 having a pitch corresponding to the distance between two subsequent output ports, the stem further being connected to a support 9. The drive means consist of a motor Ml, connected to a toothed gear 10 via a drive gear. The gear 10 could be moved along the stem 7 but is in a radially fixed position with respect to the stem by means of a notch 11. When the gear 10 makes a full turn the stem 7 will also make a turn and is moved in an axial direction through the nut 9 from one output to the following. The stem 7. is further provided with at least one cam 12 which is axially movable but radially fixed with respect to the stem and provided with a recess 13 for affecting a contact MSl, preferably a microswitch.

If a trigger impulse is supplied to the motor M1 and a rotation of the cam 12 is initiated, the contact Ms] will close the supply circuit for the motor until the cam 13 has made a full turn and the recess 13 is back at the position of the contact MSI, which means that the tap 2 has moved from one output port 4 to the subsequent port.

When separating compounds one is often only interested in collecting fractions for which the detecting signal exceeds a certain threshold value SW and all fractions having a detection signal below this level are disposed of. In the fraction characteristic of FIG. 2 such fractions are denoted 01 04. It is a purpose of the present invention to collect these waste fractions in a common vessel. For this purpose the control unit is designed so as to make it possible to determine a certain threshold value for the whole process and to generate a signal as soon as the level of the detecting signal crosses this threshold level. For this purpose the stem 7 is provided with a second cam 12a provided with a recess 13a, located e.g. in a diametrically opposed position with respect to the recess 13. The second cam 12a cooperates with a second switch M82 and the supply voltage to the motor M1 is supplied via the second switch M82 as soon as the detecting signal is below the threshold value, and the motor M1 makes a half turn of the plug 2 when the signal decreases below the threshold value. In this position the plug 2 connects the input port of the valve to one of a number of output valve ports 4a, diametrically opposed to the ports 4, the waste fractions thus being transferred to the ports 4a. The ports 4a are axially located between the output ports 4 and these ports lead to a common collecting channel 14 terminating in a common collecting container l5.

The control unit for controlling the motor Ml will now be described in detail, reference being made to FIG. 7. In the circuit diagram of FIG. 7 reference U denotes the output voltage from the detector EA, this voltage corresponding to the amplitude of the detecting signal. This voltage is supplied to a resistance R1 and is by means of an operational amplifier OP compared with a previous voltage supplied from a potentiometer P. If there exists a difference AU between the voltages of the potentiometer P and the resistance R1, this difference will be amplified and in dependence of its polarity actuate a motor M2 in either direction. The motor M2 is mechanically coupled to the potentiometer P and affects the voltage across the potentiometer in such a direction so as to eliminate the difference between the voltages across the potentiometer P and the resistance R]. This motor potentiometer thus follows the output current from the detector EA, the motor potentiometer thereby giving a much higher stability with respect to time than a corresponding capacitive network.

In order to ensure a failsafe operation of the motor the control unit could preferably be designed as illustrated by the following example, where the full amplitude of the detection signal is 100 mV and an accuracy of i 1 mV should be achieved, 1 mV being amplified to V by the operational amplifier. If a voltage difference of 1 mV gives rise to a voltage difference of 15 V at the output of the operational amplifier this voltage should form an unambiguous control signal for logic circuits connected to the output of the amplifier. The zero potential of the operational amplifier is in the middle between +15 and -l5 V and thus the zero potential of the connected logical circuits must be connected with the polarity of the operational amplifier supply voltage. The trigger circuits Trl and Tr2 are designed to switch at +10 and V respectively and the trigger circuit Tr2 is connected to the motor M2 via an inverting circuit J. The trigger circuits are further connected to a set reset flip-flop PH, and the function of the motor as well as the output signals from the flip-flop appear from the following diagram.

The motor potentiometer PM2 is mechanically connected to a micro switch S1 for determining the threshold value SW. One output of the flip-flop FFl is connected to a master slave flip-flop FFZ and the function of the flip-flops will now be described with reference to FIG. 7 and FIG. 2a. As long as the voltage difference AU is positive and consequently the curve of FIG. 2 is rising, the output Q of the flip-flop FFl has the logical value 1 and keeps this value until the voltage difference becomes negative (as at the point U' according to FIG. 2a). At this point the output 6 obtains the logical value I. This value is transferred to the master part of the flip-flop FF2 without changing the output signal of this flip-flop. (Q 0, 6 1). Only when the voltage difference becomes positive (i.e. when the containers of the fraction collector should be changed) and the flip-flop FF] again obtains the value Q l a corresponding change of condition will take place in the flip-flop FFZ and thus affect a relay RlS via' a transistor amplifier. The short impulse from the relay R18 is supplied to the contact 0 whereby the motor M1 is activated. As the motor rotates, the cam 12 will close the contact MS] thus closing the supply circuit of the motor Ml also when the relay c1 breaks and the motor will make a full turn. The impulse of the relay RlS will also reset the flip-flop FFZ via an RC network R2cl, the output 0 of the flip-flop FF2 thus being given the output signal zero.

In the above description it was supposed that the detecting signal was above the predetermined threshold value. If, however, the detecting signal is below this value, the pole c of the motor Ml will be connected to the pole b instead of the pole a by means of the mechanical coupling between the potentiometer P and the switch S1. The motor will then be connected to the power supply via the closed contact M82 and will rotate the cam a half turn when the switch M52 is broken. In order to avoid an activation of the motor due to changes of the polarity of the voltage differences AU when the detection signal is below the threshold value, the switch S1 is provided with a second contact via which the output Q of the flip-flop FF2 is maintained in a zero condition.

It is of course necessary that the power supply of the control unit is stabilized. It might also be suitable to introduce delay means in the logical circuit so as to eliminate effects e.g. from air bubbles in the tube. Suitable components for this purpose are well known per se as well as elements for changing the colour of the plotter curve, e.g. when the detecting signal passes the threshold value.

The advantages of the invention could be summarized as follows: All the fractions of interest are connected in each one container which considerably facilitates further analysis of the sample. The waste fractions are all collected in a common container. It is easy to prepare the different fraction vessels with suitable protection reagents. It will be easy to cool the samples without keeping the complete apparatus in a cool room as the vessels are not directly connected to the fraction collector but are remote from the valve by means of the tubes 5 which makes it possible to keep the containers e.g. in liquid nitrogen which is extremely important e.g. in the biological field-In the apparatus according to the invention the fractions could also be subject to a diolyzis or concentration which is often desired when dealing with proteins, macromolecular substances etc.

We claim:

1. Apparatus for transferring into separate fraction vessels a plurality of liquid fractions having a certain characteristic and being sequentially discharged from a container, the apparatus comprising:

monitoring means for monitoring substances with respect to said characteristic and generating a corresponding output signal;

a multipath valve having one input port, a number of fraction output ports and at least one waste material output port;

collecting vessels connected to said output ports;

conveying means for transferring material discharged from the container through the monitoring means to said input port of said valve;

control means for controlling said valve with respect to said signal so as to connect the input port to said waste material output port when as the level of said characteristic is below a predetermined threshold value, and to connect the input port to a fraction output port when the level of said characteristic is above said threshold level, the valve thereby sequentially connecting the fraction output ports as a rise of the characteristic is initiated. 

1. Apparatus for transferring into separate fraction vessels a plurality of liquid fractions having a certain characteristic and being sequentially discharged from a container, the apparatus comprising: monitoring means for monitoring substances with respect to said characteristic and generating a corresponding output signal; a multipath valve having one input port, a number of fraction output ports and at least one waste material output port; collecting vessels connected to said output ports; conveying means for transferring material discharged from the container through the monitoring means to said input port of said valve; control means for controlling said valve with respect to said signal so as to connect the input port to said waste material output port when as the level of said characteristic is below a predetermined threshold value, and to connect the input port to a fraction output port when the level of said characteristic is above said threshold level, the valve thereby sequentially connecting the fraction output ports as a rise of the characteristic is initiated. 